Construction Of Eukaryotic Expression Vectors Of Different HBV Gene Subtypes And Preliminary Study On HBV Virus Clearance By CRISPR / Cas9 System

Objective: To construct eukaryotic expression plasmids containing 1.3 fold complete genome of hepatitis B virus(HBV)B2?B3?C1?C2?and I1 subtypes.To explore the effect of HBV subgenotypes on the expression of HBV antigens and the cell cycles.To evaluate the inactivation effect of HBV replication by g RNA/Cas9 and optimize of an effective HBV-specific CRISPR/Cas9 system.Method: Hepatitis B virus(HBV)B2?B3?C2 subtype eukaryotic expression vectors were transiently transfected into five liver(cancer)cell lines,respectively.Culture supernatants were collected at 24,48,72,96,120,and 144 hours.HBs Ag expression was detected by ELISA;The cell cycle distribution of the five liver(cancer)cell lines with different HBV genotypes were measured by flow cytometry,respectively.To selecte the most efficient g RNA target,the HBV genome sequences of different subtypes were compared and 12 candidate g RNAs were designed and transfected into Hep G2215 cellsby using CRISPRCas9 system,and their supression effects on HBV virus genes were detected.Result: In this experiment,1.3-fold hepatitis B virus(HBV)B2?B3?C1?C2and I1 subtype recombinant plasmids were successfully transfected into five liver(cancer)cell lines,,respectively.The expression time and expression level of HBs Ag were different among the cell lines with different HBV subgenotypes.And the cell cycle distribution has been changed after transfection.In vitro expriments,the candidate g RNA/Cas9 system could suppress the expression of HBs Ag.Conclusion: Recombinant plasmids can express surface antigens and affect the cell cycle in live cell lines,and the expression time and level of HBs Ag are differ among HBV subgenotypes.The mechanism needs to be further exploited.An effective g RNA/Cas9 system that could efficiently suppress markers of viral replication was identified.